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Status |
Public on Mar 02, 2015 |
Title |
HM3-C1 |
Sample type |
SRA |
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Source name |
Primary mesothelial cell line HM3, untreated
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Organism |
Homo sapiens |
Characteristics |
cell line: HM3 cell type: primary mesothelial cells cell source: peritoneal cavity treatment: no exposure for 8 hrs
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Treatment protocol |
All cells were incubated at 37°C in 5% CO2 and grown to 80-90% confluence before addition of crocidolite asbestos fibers prepared and added (5 μg/cm2 or 75 x 10^6 μm2/cm2 for 8 h) to cell culture medium. After sterilization under ultraviolet light overnight to avoid endotoxin and microbial contamination, fibers were suspended in HBSS at 1 mg/ml, sonicated for 15 minutes in a water bath sonicator, and triturated five times through a 22-gauge needle. This suspension was added to cells in medium.
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Growth protocol |
LP9 cells were grown in 50:50 DMEM/F-12 medium containing 10% FBS, and supplemented with penicillin (50 units/ml), streptomycin (100 mg/ml), hydrocortisone (100 mg/ml), insulin (2.5 mg/ml), transferrin (2.5 mg/ml), and selenium (2.5 mg/ml); all other cells were grown in 50:50 M199:MCDB106 medium supplemented with 15% FBS, 10 ng/mL EGF, 0.4 µg/mL hydrocortisone, 50 units/mL penicillin and 100 µg/mL streptomycin.
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Extracted molecule |
total RNA |
Extraction protocol |
1 μg of total RNA was subjected to library synthesis using the TruSeq V2 RNA-Seq kit. 1 μg of total RNA was enriched for the Poly-A mRNA and reverse transcribed to double-stranded cDNA. The cDNA was end repaired, adenylated, and ligated with Illumina indexes prior to 12 cycles of PCR. Sequencing was performed using 12pM hybridization to a 2x100 paired end flow cell.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 1000 |
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Description |
2x100 paired-end sequence reads
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Data processing |
Illumina HiSeq 1000 reads were trimmed and clipped for quality control in Trimmomatic v0.27. Read quality was checked for each sample using FastQC v0.10.1. High-quality reads were then imported into TopHat v2.0.8 for alignment into BAM files. BAM files were imported into the RNA-Seq pipeline of Partek Genomics Suite, version 6.6 (Copyright 2009, Partek Inc., St. Louis, MO, USA), and RPKM counts were calculated (RefSeq release 2013-5-10). Exclusions included a minimum RPKM value of 1.0 in at least two of the 16 samples (potentially duplicates of one sample group). RPKM values were log2-transformed with an offset value of 1.0. Genome_build: Alignment was initially to the transcriptome (hg19, GRCh37.72), and then to the genome (hg19, GRCh37). Supplementary_files_format_and_content: ProcessedDataMatrix_Final.txt: Tab-delimited text file includes RPKMs (log2-transformed with an offset value of 1.0) for each sample.
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Submission date |
Dec 08, 2014 |
Last update date |
May 15, 2019 |
Contact name |
Julie Dragon |
E-mail(s) |
julie.dragon@uvm.edu
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Organization name |
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
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Department |
MMG
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Street address |
95 Carrigan Dr
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City |
Burlington |
State/province |
VT |
ZIP/Postal code |
05405 |
Country |
USA |
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Platform ID |
GPL15433 |
Series (1) |
GSE63966 |
Differential susceptibility of human pleural and peritoneal mesothelial cells to asbestos exposure |
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Relations |
BioSample |
SAMN03255847 |
SRA |
SRX800027 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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